Cell culture container and cell culture method

ABSTRACT

To provide a cell culture chamber and a cell culture method that are capable of effectively constructing an intercellular network in a culture space. A cell culture chamber ( 10 ) according to the present invention is a cell culture chamber ( 10 ) including a plurality of microchambers ( 11 ) formed on a surface thereof, characterized in that convex portions (side walls  12 ) that partition the microchambers ( 11 ) adjacent to each other are formed to prevent cells from being adhered to upper surfaces of the convex portions. Consequently, cells can be cultured exclusively within the microchambers ( 11 ), and an intercellular network can be constructed effectively.

TECHNICAL FIELD

The present invention relates to a cell culture chamber and a cellculture method.

BACKGROUND ART

A technique of using cells isolated from a tissue in testing orexamination is an essential method in the biotechnology-related fields.It is widely used in diagnosing a disease or pathological condition,searching for a new drug and evaluating the efficacy of a drug, or inanimal inspection, plant inspection, testing for environmentalpollutants, and so on. Thus, cells and the like used in thebiotechnology field have been greatly diversified.

The isolated cells are sometimes used immediately for testing, but inmany cases, the cells are cultured in a culture dish or a test tube.Various examinations are carried out using the cultured cells. Celllines in culture for use in cell culture tests are required to show drugsusceptibility and toxic reaction that are similar to those obtained ina test performed in a living body, that is, a so-called in vivo test. Inshort, it is necessary to be able to construct an intercellular networkregularly arranged on the surface of a cell culture chamber. Theintercellular network herein described refers to a state where cells canbe connected with each other and interact with each other, a form inwhich cells are accumulated to form a cell mass, or a form in whichcells are formed in a net shape. Furthermore, the cell lines in culturefor use in cell culture tests are extremely expensive, so an improvementin survival rate and proliferation rate of cells is desired.

The cell culture tests measure the effect of a drug or the like to beevaluated, by changing its amount, concentration, and the like under thesame conditions. For this reason, it is necessary that the cell culturechambers be identical in material, shape, and the like. As the cellculture chambers, a petri dish made of plastic, a petri dish made ofglass, a glass plate fixed into a chamber, a well plate, and the likeare generally used. Examples of the well plate include 6-well, 12-well,48-well, and 96-well plates or petri dishes. In general, these plateshave substantially the same overall size. As the number of wellsincreases, the size of a single well becomes smaller. A single wellcorresponds to a single culture dish. With the recent trend towardminiaturization, a 384-well plate having a number of culture dishes witha small diameter has also come to be used. Bottom surfaces of theseculture dishes have a flat plate shape, and each of the bottom surfacesis used as a culture surface.

However, the use of the conventional cell culture chamber for culturingtissue cells causes the cells to be thinned into a form with noorientation. Additionally, the cells are randomly arranged on thesurface of the cell culture chamber, so intercellular networks crosseach other in a complicated manner. This causes a problem of beingincapable of reproducing cell functions in vivo.

As methods for solving the above-mentioned problem and culturing cellsin three dimensions, there are disclosed a method for culturing cellsutilizing a cell culture chamber having a size on the order of severalhundreds of μm (see Patent Document 1), a method for culturing cellsutilizing micropatterns including a cell placement section and a flowchannel (see Patent Document 2), and the like.

[Patent Document 1] Japanese Unexamined Patent Application PublicationNo. 2004-154027

[Patent Document 2] Japanese Unexamined Patent Application PublicationNo. 2006-191809

DISCLOSURE OF INVENTION Technical Problem

In both Patent Documents 1 and 2, convex portions for partitioning aspace for culturing cells are formed. In Patent Document 1, however, thewidth of the upper surface of each convex portion is about twice orthree times the size of a cell. This causes a problem that cells areadhered to the upper surface and an intercellular network is notconstructed effectively in a culture space. On the other hand, in PatentDocument 2, the width of each convex portion is smaller than the size ofa cell, but the height of each convex portion is lower. This causes aproblem that cells run on the convex portions and an intercellularnetwork is not constructed effectively in the culture space.

The present invention has been made to solve the above-mentionedproblems, and therefore has an object to provide a cell culture chamberand a cell culture method that are capable of effectively constructingan intercellular network in a culture space.

Technical Solution

A cell culture chamber according to the present invention is a cellculture chamber including a plurality of microchambers formed on asurface thereof, characterized in that convex portions that partitionthe microchambers adjacent to each other are formed to prevent cellsfrom being adhered to upper surfaces of the convex portions. The convexportions may have a multi-stage structure to prevent cells from beingadhered to an upper surface of each stage. Further, it is preferablethat the upper surfaces of the convex portions have a short side widthof 0.5 to 15 μm and the convex portions have a height equal to or morethan three times the short side width. The convex portions preferablyhave a height of 30 to 300 μm.

Further, in 50% or more of an upper portion in a height direction of theside walls with a horizontal plane of each of the microchambers as areference surface, an angle formed between the horizontal plane and eachside surface of the side walls is preferably 80° to 90°.

Further, each of the microchambers preferably has a bottom surface areaof 6.25×10⁻⁴ mm² to 0.563 mm². In the case where cultured cells areliver cells, a major axis of the bottom surface is preferably 1 to 1.5times a minor axis thereof. Meanwhile, in the case of evaluatingmigration properties of cells, the major axis of the bottom surface ispreferably 1.5 to 50 times the minor axis thereof.

Furthermore, it is preferable that the microchambers communicate with atleast one adjacent microchamber and an opening therefor have a bottomsurface width of 1 μm to 25 μm.

Further, it is preferable that an area having the microchambers formedtherein be subjected to surface treatment and an integrated layer filmformed by the surface treatment have two or more layers including atleast one layer of an inorganic film and at least one layer of anorganic film. Moreover, the area is preferably transparent.

A cell culture method according to the present invention is a method forinjecting cells into the microchambers formed in the cell culturechamber, and for culturing the cells in the above-mentioned cell culturechamber. The cells are preferably selected from liver cells, fat cells,osteoblasts, pulp cells, cartilage cells, stem cells, nerve cells, andcardiac muscle cells.

ADVANTAGEOUS EFFECTS

According to the present invention, it is possible to provide a cellculture chamber and a cell culture method that are capable ofeffectively constructing an intercellular network in a culture space.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plane view showing the structure of a cell culture chamberaccording to an embodiment of the present invention;

FIG. 2 is a sectional view showing the structure of the cell culturechamber according to an embodiment of the present invention;

FIG. 3 is a plane view showing the structure of a cell culture chamberaccording to an embodiment of the present invention;

FIG. 4 is a sectional view showing the structure of the cell culturechamber according to an embodiment of the present invention;

FIG. 5 is a plane view showing the structure of a cell culture chamberaccording to an embodiment of the present invention;

FIG. 6 is a sectional view showing the structure of the cell culturechamber according to an embodiment of the present invention;

FIG. 7 is an optical microscope image of cultured cells in a cellculture chamber according to Example 1;

FIG. 8 is an optical microscope image of cultured cells in the cellculture chamber according to Example 1;

FIG. 9 is an optical microscope image of cultured cells in a cellculture chamber according to Comparative Example 1;

FIG. 10 is an optical microscope image of cultured cells in the cellculture chamber according to Comparative Example 1;

FIG. 11 is a plane view showing the structure of a cell culture chamberaccording to an embodiment of the present invention;

FIG. 12 is a sectional view showing the structure of the cell culturechamber according to an embodiment of the present invention; and

FIG. 13 is an optical microscope image of cultured cells in a cellculture chamber according to Example 2.

EXPLANATION OF REFERENCE

-   10 CELL CULTURE CHAMBER-   11 MICROCHAMBER-   12 SIDE WALL-   121 FIRST SIDE WALL-   122 SECOND SIDE WALL-   13 OPENING

BEST MODES FOR CARRYING OUT THE INVENTION

A cell culture chamber according to the present invention has aconcave-convex pattern, i.e., a plurality of microchambers or culturespaces formed therein. The width and height of side walls (convexportions) for partitioning the microchambers are optimized, therebymaking it possible to culture cells exclusively within the microchambersand construct an intercellular network effectively.

The dimensions of the microchambers each surrounded by the side wallshave to be set within the optimum range for culturing cells. If thebottom surface area of each microchamber is too large, cells are thinlyelongated and fail to show a three-dimensional structure, as in theculture on a flat plate. If, on the other hand, the bottom surface areaof each microchamber is too small, it cannot accommodate cells.Accordingly, the dimensions of the space structure are preferably in arange capable of containing one or a plurality of cells according tocell species to be cultured. In the case of forming a cell mass in whicha plurality of cells is accumulated, the dimensions are preferably in arange capable of containing the cell mass.

It is also necessary to set the side walls of the microchambers withinthe optimum range for culturing cells. If the width of each side wall istoo large, cells are adhered to the upper surface of the side wall, andthus such side wall is unsuitable for culture. If the width of each sidewall is too small, the production thereof becomes difficult. If theheight of each side wall is too low, cells run on the side wall, andthus such side wall is unsuitable for culture. If the height of eachside wall is too high, the production thereof is difficult and materialdiffusion becomes difficult, leading to a deterioration of the cultureenvironment.

In addition, openings are formed in the side walls to obtain a structurein which the plurality of microchambers communicates with each other,thereby making it possible to supply oxygen and nutrients to cells andremove waste products from the cells effectively. Note that the heightof the side walls, the dimensions of the microchambers, and the width ofthe openings are appropriately set according to cell species to becultured, thereby enabling application to various culture systems.

Hereinafter, embodiments of the present invention will be described.Note that the present invention is not limited to embodiments describedbelow. To clarify the explanation, the following description and thedrawings are simplified as appropriate.

Embodiments

The structure of a cell culture chamber according to an embodiment ofthe present invention will be described with reference to FIGS. 1 and 2.FIG. 1 is a plane view showing the structure of the cell culture chamberaccording to this embodiment, and FIG. 2 is a sectional view taken alongthe line II-II of FIG. 1. As shown in FIG. 1, a cell culture chamber 10includes microchambers 11, side walls 12, and openings 13. The pluralityof side walls 12 is formed in a net shape on the culture surface of thecell culture chamber 10, and spaces surrounded by the side walls 12correspond to the microchambers 11. Additionally, each of the openings13 is formed at a central portion of each side of the side walls 12which are formed on four sides of each of the microchambers 11 having arectangle shape.

FIG. 1 shows a width “a” of the bottom surface of each of themicrochambers 11, a width “b” and a height “c” of each of the side walls12 for partitioning the microchambers 11, and a width “d” of each of theopenings 13 for allowing communication between the microchambers 11adjacent to each other. Here, it is necessary that 0.5 μm≦b≦15 μm andc/b≧3 be satisfied. If the height “b” of each of the side walls 12exceeds 15 μm, cells are adhered to the upper surfaces of the sidewalls, and thus such side walls are unsuitable for culture. If, on theother hand, the width “b” of each of the side walls 12 is less than 0.5μm, the production thereof becomes difficult. If the height of each ofthe side walls is too low, cells run on the side walls, and thus suchside walls are unsuitable for culture. If the height “c” of each of theside walls 12 is less than twice the width “b” of each of the side walls12, cells to be cultured in the microchambers 11 run on the side wallsand move to the adjacent microchambers 11. Additionally, the height “c”of each of the side walls 12 is preferably within a range of 30 μm to300 μm. Specifically, in the case of forming a cell mass having anequivalent diameter of 100 μm, the height “c” of each of the side walls12 is preferably 50 μm to 150 μm. Here, if the height “c” of each of theside walls is too high, the production thereof is difficult and materialdiffusion becomes difficult, leading to a deterioration of the cultureenvironment. The side walls 12 may have a multi-stage shape.

The bottom surface shape of each of the microchambers 11 is notparticularly limited, and various shapes other than a square, a circle,and a polygon can be employed. This bottom surface area is preferably6.25×10⁻⁴ mm² to 0.563 mm². Specifically, in cell culture forreproducing a liver function in vivo, this bottom surface area ispreferably 0.01 mm² to 0.1 mm². In this case, the major axis of thebottom surface is preferably 1 to 1.5 times the minor axis thereof. Anisotropic shape is more preferably used. If a square is employed, forexample, in the case of forming a cell mass having an equivalentdiameter of 100 μm, the length of one side thereof is preferably 100 μmto 300 μm.

Further, in the culture for orienting nerve cells in order to reproducea neural network in vivo, a microchamber having a rectangle shape and anopening can be employed. For example, it is preferable that theshort-side width of the microchamber be 20 μm and the length of the longside thereof be equal to or greater than 100 μm. That is, the major axisof the bottom surface is preferably equal to or more than five times theminor axis thereof.

In the case of evaluating migration properties of cells in order toexamine the functions of cells in vivo, a rectangle shape can beemployed. For example, it is preferable that the short side width of themicrochamber be 15 μm and the length of the long side thereof be 22.5 to750 μm. That is, the major axis of the bottom surface is preferably 1.5to 50 times the minor axis thereof.

An angle formed between the horizontal plane and the side wall 12 ofeach of the microchambers 11 should be set to an angle at which cellsare prevented from running on the microchambers. Accordingly, 50% ormore of an upper portion of a side surface preferably has an angle of 80to 90°, and more preferably, 85° to 90°.

The width “d” of each of the openings 13 for allowing communicationbetween the microchambers 11 adjacent to each other is preferably set toa width in which cells are prevented from moving from the microchamber11, in which the cultured cell is first seeded, to the adjacentmicrochamber 11. When the equivalent diameter of the cultured cell is 20μm, for example, the width is preferably 5 to 15 μm. In addition, likethe concave-convex pattern shown in FIGS. 11 and 12, each of theopenings 13 is not necessarily formed at the center of each of themicrochambers 11, but may be formed at a corner portion (corner portionof a rectangular). Here, FIG. 11 is a plane view showing the structureof another cell culture chamber according to this embodiment, and FIG.12 is a sectional view taken along the line XI-XI of FIG. 11. Note thatthe openings 13 are not necessarily formed. As shown in FIGS. 3 and 4,the four sides of each of the microchambers 11 may be entirelysurrounded by the side walls 12. Here, FIG. 3 is a plane view showingthe structure of another cell culture chamber according to thisembodiment, and FIG. 4 is a sectional view taken along the line IV-IV ofFIG. 3.

Also, as shown in FIGS. 5 and 6, the microchambers 11 having a circularshape may be entirely surrounded by first side walls 121, and secondside walls 122 may be formed on the first side walls 121. That is, thefirst side walls 121 and the second side walls 122 constitute the sidewalls (convex portions) 12 of a multi-stage structure. Here, FIG. 5 is aplane view showing the structure of another cell culture chamberaccording to this embodiment, and FIG. 6 is a sectional view taken alongthe line VI-VI of FIG. 5.

A method for forming the concave-convex pattern on the cell culturechamber of the present invention is not particularly limited, butmethods such as transfer molding using a mold, three-dimensionalstereolithography, precision machining, wet etching, dry etching, laserprocessing, and electrical discharge machining may be employed. It ispreferable to appropriately select these production methods in view ofthe intended use, required processing accuracy, costs, and the like ofthe cell culture chamber.

As a specific example of the transfer molding method using a mold, amethod for forming the concave-convex pattern by resin molding using ametal structure as a mold may be employed. This method is preferredbecause it is capable of reproducing the shape of the metal structure ona resin as the concave-convex pattern with a high transcription rate,and because the raw material cost can be reduced by using ageneral-purpose resin material. Such a method using a mold of a metalstructure is superior in terms of low cost and achieving satisfactorilyhigh dimensional accuracy.

As methods of producing the metal structure, for example, platingtreatment, precision machining, wet etching, dry etching, laserprocessing, and electrical discharge machining on a resist patternproduced by photolithography or a resin pattern produced bythree-dimensional stereolithography may be employed. The methods may beappropriately selected in view of the intended use, required processingaccuracy, costs, and the like.

As methods of forming the concave-convex pattern on a resin using themetal structure, which is obtained as described above, as a mold,injection molding, press molding, monomer casting, solvent casting, hotembossing, or roll transfer by extrusion molding may be employed, forexample. It is preferable to employ injection molding in view of itsproductivity and transcription property.

Materials for forming the cell culture chamber of the present inventionare not particularly limited as long as the materials haveself-supporting properties. For example, synthetic resin, silicon, orglass may be employed. A transparent synthetic resin is preferably usedas a material in view of costs and cell visibility under microscopicalobservation. Examples of the transparent synthetic resin include acrylicresins such as polymethylmethacrylate or methyl methacrylate-styrenecopolymer, styrene resin such as polystyrene, olefin resin such ascycloolefin, ester resins such as polyethylene terephthalate andpolylactic acid, silicone resin such as polydimethylsiloxane, andpolycarbonate resin. These resins may contain various additives such ascolorant, dispersing agent, and thickening agent, unless thetransparency is impaired.

In the cell culture chamber of the present invention, surface treatmentmay be performed on the surface side of the concave-convex pattern and amodified layer and/or a coating layer may be formed for the purpose ofimproving the hydrophilic properties, biocompatibility, cellularaffinity, and the like of the chamber surface. A method for forming themodified layer is not particularly limited unless a method with whichthe self-supporting properties are impaired and a method causing extremesurface roughness of 10 μm or more are employed. Methods, for example,chemical treatment, solvent treatment, chemical treatment such asintroduction of a graft polymer by surface graft polymerization,physical treatment such as corona discharge, ozone treatment, or plasmatreatment may be employed. In addition, though a method for forming thecoating layer is not particularly limited, methods, for example, drycoating such as sputtering or vapor deposition and wet coating such asinorganic material coating or polymer coating may be employed. In orderto inject a culture solution without mixing air bubbles therein, it isdesirable to impart the hydrophilic properties to the surface of theconcave-convex pattern. As a method for forming a uniform hydrophilicmembrane, inorganic vapor deposition is preferably employed.

When the cellular affinity is taken into consideration, it is morepreferable to coat cytophilic proteins such as collagen and fibronectin,for example. In order to coat a collagen aqueous solution or the likeuniformly, it is preferable to perform the coating after theabove-mentioned hydrophilic membrane is formed. In cell culture, ingeneral, it is desirable to culture cells on an extracellular matrixsurface by replicating the in vivo environment. Accordingly, it isparticularly preferable to dispose an organic film made of extracellularmatrix suitable for cultured cells after an inorganic hydrophilicmembrane is uniformly formed as described above.

In a cell culture method of the present invention, an appropriate numberof cells need to be seeded so that the cells are arranged exclusivelywithin the microchambers for culturing cells and morphologies andfunctions similar to those in vivo are developed within the space. Acell seeding density of 1.0×10⁴ to 1.0×10⁶ cells/cm² is preferably used.When each microchamber is a square which is 200 μm on a side, forexample, a cell seeding density of 5.0×10⁴ to 5.0×10⁵ cells/cm² ispreferably used. Under such conditions, a cell mass having a diameter of30 to 200 μm can be obtained.

Mode for the Invention 1

Next, examples of the cell culture chamber according to the presentinvention will be described, but the present invention is not limited tothese examples.

<Preparation of Liver Cells>

Liver cells of a primary rat for use in culture were prepared in amanner as described below. A surflow indwelling needle was inserted intothe portal vein of a 6-week-old Wistar rat, and blood removal wasperformed by causing an EDTA-containing solution to flow. Then, acollagenase solution was perfused. After that, the liver treated by thecollagenase solution was immersed in a culture solution, and the cellswere dispersed by pipetting using a measuring pipette. The cellsuspension was cleaned three times to remove cells other than the cells,and isolated cells were used for culture.

<Culture Method>

A culture solution for use in culture was prepared in a manner asdescribed below.

A culture medium of DMEM/F12 was added with 10% fetal bovine serum, 1μg/ml insulin, 1×10⁻⁷ M dexamethasone, 10 mM nicotinamide, 2 mML-glutamine, 50 μm β-mercaptoethanol, 5 mM HEPES, 59 μg/ml penicillin,100 μg/ml streptomycin, 25 ng/ml HGF, and 20 ng/ml EGF.

On a base material having a concave-convex pattern, liver cells wereseeded at a density of 1.0×10⁵ cells/cm² and were cultured for apredetermined period of time with 5% CO₂ and at 37° C. Further, 0.5 mLfresh culture medium having the same composition was used, and theculture medium was changed every day or every two days.

Example 1

A pattern which has the shape of the concave-convex pattern as shown inFIG. 3 and which has dimensions of a=100 um, b=10 um, and c=50 um wasproduced by photolithography, and Ni electrolytic plating was carriedout to obtain a mold having a corresponding concave-convex shape.Pattern transcription was performed on polystyrene by hot embossing withthe mold, and a resin base material having the above-mentioneddimensions was produced. A silicon dioxide film was formed with athickness of 100 nm on the surface of the resin base material by vacuumdeposition, and γ-ray sterilization was carried out to obtain the basematerial having the concave-convex pattern. Liver cells were cultured onthe concave-convex base material.

Comparative Example 1

A pattern which has the shape of the concave-convex pattern as shown inFIG. 3 and which has dimensions of a=100 um, b=20 um, and c=50 um wasproduced by photolithography, and Ni electrolytic plating was carriedout to obtain a mold having a corresponding concave-convex shape.Pattern transcription was performed on polystyrene by hot embossing withthe mold, and a resin base material having the above-mentioneddimensions was produced. A silicon dioxide film was formed with athickness of 100 nm on the surface of the resin base material by vacuumdeposition, and γ-ray sterilization was carried out to obtain the basematerial having the concave-convex pattern. Liver cells were cultured onthe concave-convex base material.

FIG. 7 is an optical microscope photograph showing a state after cellswere seeded under the conditions of Example 1 and the cells werecultured for four hours. FIG. 8 is an optical microscope photographshowing a state after the cells were cultured for four days. The cellsare not adhered to the upper surfaces of the convex portions thatpartition the culture spaces, and the cells are successfully culturedexclusively within the concave portions that are original culturespaces. As a result, an intercellular network could be constructed inthe culture space and the functions similar to those in vivo could bedeveloped.

FIG. 9 is an optical microscope photograph showing a state after cellswere seeded under the conditions of Comparative Example 1 and the cellswere cultured for four hours. FIG. 10 is an optical microscopephotograph showing a state after the cells were cultured for four days.The cells are adhered also to the upper surfaces of the convex portionsthat partition the culture spaces, and were cultured thereon.Additionally, the adjacent culture spaces could not be distinctlypartitioned, and the cells could not be cultured exclusively within theculture spaces. As a result, an intercellular network could not beconstructed in the culture spaces, and the functions similar to those invivo could not be developed.

Example 2

A pattern which has the shape of the concave-convex pattern as shown inFIGS. 11 and 12 and which has dimensions of a=80 um, b=15 um, and c=50um was produced by photolithography, and Ni electrolytic plating wascarried out to obtain a mold having a corresponding concave-convexshape. Pattern transcription was performed on polystyrene by hotembossing with the mold, and a resin base material having theabove-mentioned dimensions was produced. A silicon dioxide film wasformed with a thickness of 100 nm on the surface of the resin basematerial by vacuum deposition, and γ-ray sterilization was carried outto obtain the base material having the concave-convex pattern. Livercells were cultured on the concave-convex base material.

FIG. 13 is an optical microscope photograph showing a state after cellswere seeded under the conditions of Example 2 and the cells werecultured for four hours. The cells are not adhered to the upper surfacesof the convex portions that partition the culture spaces, and the cellsare successfully cultured exclusively within the concave portions thatare original culture spaces. As a result, an intercellular network couldbe constructed in the culture spaces and the functions similar to thosein vivo could be developed.

1. A cell culture chamber comprising a plurality of microchambers formedon a surface thereof, wherein convex portions that partition themicrochambers adjacent to each other are formed to prevent cells frombeing adhered to upper surfaces of the convex portions.
 2. The cellculture chamber according to claim 1, wherein the convex portions have amulti-stage structure to prevent cells from being adhered to an uppersurface of each stage.
 3. The cell culture chamber according to claim 1,wherein each of the upper surfaces of the convex portions has a shortside width of 0.5 to 15 μm, and each of the convex portions has a heightequal to or more than three times the short side width.
 4. The cellculture chamber according to claim 3, wherein each of the convexportions has a height of 30 to 300 μm.
 5. The cell culture chamberaccording to claim 1, wherein, in 50% or more of an upper portion in aheight direction of the convex portions comprising a bottom surface ofeach of the microchambers as a reference surface, an angle formedbetween the bottom surface and each side surface of the convex portionsis 80° to 90°.
 6. The cell culture chamber according to claim 1, whereineach of the microchambers has a bottom surface area of 6.25×10⁻⁴ mm² to0.563 mm².
 7. The cell culture chamber according to claim 1, wherein abottom surface of each of the microchambers has a major axis which is 1to 1.5 times a minor axis thereof.
 8. The cell culture chamber accordingto claim 1, wherein a bottom surface of each of the microchambers has amajor axis which is 1.5 to 50 times a minor axis thereof, and migrationproperties of cells are evaluated.
 9. The cell culture chamber accordingto claim 1, wherein the microchambers communicate with at least oneadjacent microchamber.
 10. The cell culture chamber according to claim9, wherein an opening for allowing the microchambers to communicate withthe at least one adjacent microchamber has a width of 1 to 25 μm. 11.The cell culture chamber according to claim 1, wherein an area havingthe micropatterns formed therein is subjected to surface treatment. 12.The cell culture chamber according to claim 11, wherein an integratedlayer film formed by the surface treatment has two or more layerscomprising at least one layer of an inorganic film and at least onelayer of an organic film.
 13. The cell culture chamber according toclaim 1, wherein an area having the micropatterns formed therein istransparent.
 14. A cell culture method comprising injecting cells intothe microchambers formed in the cell culture chamber according to claim1, and culturing the cells.
 15. The cell culture method according toclaim 14, wherein the cells are selected from the group consisting ofliver cells, fat cells, osteoblasts, pulp cells, cartilage cells, stemcells, nerve cells, and cardiac muscle cells.
 16. The cell culturechamber according to claim 3, wherein the convex portions have amulti-stage structure to prevent cells from being adhered to an uppersurface of each stage.
 17. The cell culture chamber according to claim3, wherein, in 50% or more of an upper portion in a height direction ofthe convex portions comprising a bottom surface of each of themicrochambers as a reference surface, an angle formed between the bottomsurface and each side surface of the convex portions is 80° to 90°. 18.The cell culture chamber according to claim 3, wherein each of themicrochambers has a bottom surface area of 6.25×10⁻⁴ mm² to 0.563 mm².19. The cell culture chamber according to claim 3, wherein a bottomsurface of each of the microchambers has a major axis which is 1 to 1.5times a minor axis thereof.
 20. The cell culture chamber according toclaim 3, wherein a bottom surface of each of the microchambers has amajor axis which is 1.5 to 50 times a minor axis thereof, and migrationproperties of cells are evaluated.